Oxidative stress begins with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as a part of normal cellular function. There are multiple cellular sources of ROS generation but the most significant ones are the mitochondria electron transport complexes I and III, P450 enzymes within the endoplasmic reticulum, and membrane bound NADPH oxidase. ROS production by each of these sources can be stimulated by cytokines, inflammation, viral proteins, and other mechanisms, like chemotherapy drugs, ischemia-reperfusion, and iron and copper overload. Importantly, these processes initially generate the free-radical superoxide (.O2−) which is sequentially reduced to form hydrogen peroxide, hydroxyl radical and, ultimately, water. Under conditions of high oxidative stress and consequently high production of superoxide, these reactive intermediates, however, readily interact with other molecules to form secondary harmful ROS, such as lipid peroxidation products and peroxynitrite (Singal et al., Liver Int. 2011; 31:1432-1448). This indicates the importance of keeping the cellular amount of superoxide under tight control. Under normal conditions this is achieved by superoxide dismutases (SODs). Although SODs have the fastest reaction rate of known enzymes, under conditions of high oxidative stress, these enzymes may be outcompeted and even irreversible irreversibly inactivated by ROS and RNS. This in turn, opens up for therapeutic use of low molecular drugs that mimic the SOD enzymes, i.e., the so-called SOD mimetics, to combat pathological oxidative stress.
Short-lived but highly reactive oxygen-derived free radicals have long been known to participate in pathological tissue damage, especially during treatment with cytotoxics/cytostatics and radiotherapy in cancer patients (Towart et al., Arch Pharmacol 1998; 358 (Suppl 2):R626, Laurent et al., Cancer Res 2005; 65:948-956, Karlsson et al., Cancer Res 2006; 66:598, Alexandre et al., J Natl Cancer Inst 2006; 98:236-244, Doroshow, J Natl Cancer Inst 2006; 98:223-225, Citrin et al, Oncologist, 2010; 15:360-371, Kurz et al., Transl Oncol 2012; 5:252-259), acetaminophen-induced liver failure (Bedda et al., J Hepatol 2003; 39:765-772; Karlsson, J Hepatol 2004; 40:872-873), in ischemic heart disease (Cuzzocrea et al., Pharmacol Rev 2001; 53:135-159) and in various neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis (Knight, Ann Clin Lab Sci. 1997; 27:11-25). Overproduction of oxygen-derived free radicals is also implicated in pathological conditions of iron overload (Rachmilewitz et al., Ann N Y Acad Sci. 2005; 1054:118-23), for example, in thalassemia, sickle cell anemia and transfusional hemosiderosis. Oxygen-derived free radicals are also implicated in hepatitis-induced liver cirrhosis (Farrell et al., Anat Rec 2008; 291:684-692) and in noise-induced hearing loss (Wong et al., Hear Res 2010; 260:81-88).
The use of dipyridoxyl based chelating agents and their metal chelates and certain manganese-containing compounds, in particular manganese chelates, in medicine is known. See EP 0910360, U.S. Pat. No. 6,147,094, EP 0936915, U.S. Pat. No. 6,258,828, EP 1054670, U.S. Pat. No. 6,310,051, EP 1060174, and U.S. Pat. No. 6,391,895, for example, which disclose that certain chelating agents, in particular dipyridoxyl chelating agents, and their metal chelates, are effective in treating or preventing anthracycline-induced cardiotoxicity, radiation-induced toxicity, ischemia-reperfusion-induced injuries, and paracetamol (acetaminophen) induced liver failure, or from a more general point of view, every pathological condition caused by the presence of oxygen-derived free radicals, i.e., oxidative stress, in humans and animals. Furthermore, the dipyridoxyl compound mangafodipir (MnDPDP) has in addition and surprisingly been found to possess cytotoxic effects against cancer cells (EP 16944338). However, as described in WO 2009/078794 A1 and in Kurz et al., 2012, this is an inherent property of fodipir (DPDP) alone or its dephosphorylated counterparts, DPMP and PLED, and not of the metal complex MnDPDP or its dephosphorylated counterparts, MnDPMP and MnPLED.
One of the MnPLED-derivatives, namely manganese N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid (Manganese DiPyridoxyl DiPhosphate; MnDPDP), also known as mangafodipir, is approved for use as a diagnostic MRI contrast agent in humans. Interestingly, mangafodipir has also been shown to protect mice against serious side effects of several cytotoxic/cytostatic drugs (doxorubicin, oxaliplatin, 5-fluorouracil and paclitaxel), without interfering negatively with the anticancer effects of these drugs (Towart et al., 1998, Laurent et al., 2005, Karlsson et al., 2006, Alexandre et al., 2006, Doroshow, 2006, Kurz et al., 2012). Mangafodipir has been tested in one colon cancer patient going through palliative treatment with a combination of folinate, 5-fluorouracil and oxaliplatin (Yri et al., Acta Oncol. 2009; 48:633-635). The preclinical data and the results from this single patient were so promising that clinical testing in cancer patients has started. When it comes to the most troublesome side effect of oxaliplatin, namely oxaliplatin-induced sensory neurotoxicity, no preclinical data exist, to the best of our knowledge, showing protective effects of mangafodipir (Karlsson et al., Transl Oncol. 2012; 5:32-38). Yri et al., 2009, described that the patient received 15 full-doses of “Nordic FLOX”. In 14 of the cycles, the patient received pretreatment with mangafodipir. The patient received an accumulated dose of 1275 mg/m2 oxaliplatin, which is a dose likely to give neurotoxic symptoms. No neurotoxic symptoms were detected, except during the fifth cycle when mangafodipir was deliberately left out and the patient experienced peripheral sensory neuropathy. This suggests that mangafodipir may protect against peripheral neurotoxicity. After five cycles, the performance status for the patient was drastically improved, and the demand for analgesics was significantly reduced. Neutropenia did not occur during any of the chemotherapy cycles.
A first feasibility study (MANFOL I) has been completed and positive results, including myeloprotective effects, have been reported to the Swedish Medical Agency and have been published (Karlsson et al., 2012).
Mangafodipir has also been described to protect mice against acetaminophen-induced acute liver failure in mice (ALF) (Bedda et al., 2003; Karlsson, 2004). ALF is characterized by massive hepatocyte cell death, a condition caused by glutathione depletion, oxygen-derived free radicals and mitochondrial damage.
Mangafodipir is a pro-drug in the sense that it probably has to be metabolized into N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (MnPLED) before it can exert cytoprotective effects during in vivo conditions (e.g., see Karlsson et al., Acta Radiol 2001; 42:540-547; Kurz et al., 2012). Manganese is an essential as well as potentially neurotoxic metal. It has been known for many years that under conditions of chronic exposure to high levels of manganese, a syndrome of extrapyramidal dysfunction similar to Parkinson's syndrome, although clinically a different disease entity, frequently occurs (see Scheuhammer & Cherian, Arch Environm Contam Toxicol 1982; 11:515-520). When a diagnostic MR imaging dose of mangafodipir is intravenously injected into humans, about 80% of the administered manganese is released (Toft et al., Acta Radiol 1997; 38:677-689). Release of paramagnetic manganese is in fact a prerequisite for the diagnostic MR imaging properties of mangafodipir (Wendland, N M R Biomed 2004; 17:581-594). Elizondo et al., 1991 (Radiology 1991; 178:73-78) stated that the fodipir moiety binds to the pyridoxyl 5′ phosphate receptor on hepatocytes and ensures a high intracellular concentration of mangafodipir in the liver. This hypothesis was recently also suggested in a paper by Coriat et al., (PLoS One 2011; 6:1-6, e27005). This is a nice hypothesis but unfortunately an unproven and a very unlikely one, which fell out of fashion shortly after it had been presented. When mangafodipir is injected intravenously (i.v.) about 80% of the metal complex falls apart (Toft et al., Radiol 1997), and at every equimolar Mn dose, MnCl2 has an equal or better liver MR imaging contrast efficacy than mangafodipir (Southon et al., Acta Radiol 1997). Furthermore, after injection of mangafodipir almost all fodipir is recovered in the urine (the major part of it as PLED), whereas most manganese is recovered in the feces (Hustvedt et al., Acta Radiol 1997; 38:690-699). On the other hand, the therapeutic effects of mangafodipir (MnDPDP) and its dephosphorylated counterparts MnDPMP (N,N′-dipyridoxylethylenediamine-N,N′-diacetate-5-phosphate) and MnPLED depend on the intact metal complex (Brurok et al., Biochem Biophys Res Commun. 1999; 254:768-721, Karlsson et al 2001; 42:540-547).
PLED-derivatives mimic the mitochondrial enzyme manganese superoxide dismutase (MnSOD) (Brurok et al., 1999). MnSOD protects the mammalian cell from the superoxide radical, a byproduct from oxygen metabolism, which is produced in fairly high amounts during normal aerobic conditions; no mammalians survive without a functional MnSOD. MnSOD has the fastest turnover number (reaction rate with its substrate) of any known enzyme (>109M−1 s−1) (Fridovich, J Exp Biol. 1998; 201:1203-1209). Low molecular weight MnSOD mimetics may have turnover rates close to that of native MnSOD (Cuzzocrea et al., 2001). Interestingly, physiological buffers containing transition metals like manganese may have similar high turnover numbers (Culotta et al., Biochim Biophys Acta. 2006; 1763:747-758). However, the importance of native SOD enzymes is consistent with a selection process favoring organisms that elaborate a means of localizing transition metal catalyst for superoxide dismutation to parts of the cell where there is a high need for such dismutation, e.g., mitochondria. Furthermore, results from myocardial ischemia-reperfusion in anaesthetized pigs inevitably show that the intact MnPLED, but not manganese per se, protects against oxidative stress, seen as reduction in infarct size (Karlsson et al., 2001). Effective inactivation of superoxide is essential in preventing generation of very devastating hydroxyl radicals and peroxynitrite (Cuzzocrea et al., 2001). During pathological oxidative stress, the formation of superoxide radicals often exceeds the endogenous capacity for inactivation. Furthermore, superoxide stimulates production of peroxynitrite which nitrates endogenous MnSOD. This protein is nitrated by peroxynitrite in Tyr-34 (Radi, Proc Natl Acad Sci USA 2004; 101:4003-4008). Once nitrated, MnSOD looses its enzymatic activity, an event favoring the accumulation of superoxide and superoxide-driven damage (Muscoli et al., Br J Pharmacol 2003; 140:445-460).
Recent results indicate that MnSOD inactivation by nitration is an early event in paracetamol-induced hepatic toxicity (Agarwal et al., J Pharmacol Exp Ther 2011; 337:110-116). Old results, in addition, indicate that nitration and inactivation of MnSOD are involved in chronic rejection of transplanted kidneys in humans (MacMillan-Crow et al., Proc Natl Acad Sci USA 1996; 93:11853-11858). It may also be relevant to note that actin, which can constitute 5% or more of the cell protein, is heavily nitrated in sickle cell anemia and that the extent of nitration observed is sufficient to induce cytoskeletal polymerization (Radi, 2004). Circulating levels of 3-nitrotyrosine may in addition serve as a biomarker to assess atherosclerosis risks. Furthermore, in addition to atherosclerosis, peroxinitrite and 3-nitrotyrosine are believed to be involved in myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis (Beckman et al., Am J Physiol 1996; 271:C1424-C1437) and diabetes (Fönstermann et al, Br J Pharmacol. 2011; 164:213-223).
Impaired antioxidant defense mechanisms, including reduced SOD activity, and a subsequently increased production of peroxynitrite, may be an important factor in the pathogenesis of non-alcoholic steatohepatitis (NASH) (Koruk et al., Ann Clin Lab Sci. 2004; 34:57-62). A major epidemiological and clinical association between either hepatitis B or hepatitis C virus infections and the development of chronic hepatitis and the appearance of hepatocellular carcinoma is evident. Interestingly, peroxynitrite-induced tyrosine-nitration is markedly increased in patients with chronic viral hepatitis (Garcia-Monzon et al., J Hepatol. 2000; 32:331-338). Currently, the generally cited mechanism of pathology development in Wilson's disease involves oxidative damage due to copper overload. Generation of reactive oxygen species (ROS) as well as lipid oxidation and DNA damage has been detected in the liver, particularly at the advanced stages of this disease (Burkhead et al., Biometals 2011; 24:455-466).
MnPLED-derivatives are not targets for peroxynitrite and addition of exogenous MnPLED-derivatives may in such situations re-establish the protective potential. PLED-derivatives are in addition strong iron binders, as described in EP 1054670, U.S. Pat. No. 6,310,051 and by Rocklage et al., (Inorg Chem 1989; 28:477-485), and some MnPLED-derivatives may have catalase and glutathione reductase activities (Laurent et al., 2005), which may further increase their antioxidant capacity.
For diagnostic imaging use and other sporadic use, dissociation of manganese from mangafodipir presents no major toxicological problem. Due to uptake into CNS, however, for more frequent use, for example in therapeutic methods, accumulated manganese toxicity may represent a serious neurotoxicological problem (Crossgrove et al, NMR Biomed. 2004; 17:544-53). Thus, for more frequent therapeutic use, compounds that readily dissociate manganese should be avoided and there is a need to develop means for obtaining desirable therapeutic effects while reducing the undesirable side effects associated with such therapeutic use.